Of the varied diseases that afflict mankind, many involve abberrations in either the basic biosynthetic pathway or the salvage and interconversion pathways for purine nucleotides. The understanding of the regulation of the purine biosynthetic pathway may ultimately lead to the control and treatment of such conditions as gout, Lesch-Nyhan syndrome and severe combined immunodeficiency syndrome. The bacterium Escherichia coli K12 has been chosen as a model system as it offers several advantages over the more complicated eucaryotic systems for the study of potentially intricate and involved control systems. Purines nucleotides play a variety of roles in the cell as they are involved as a major constituent of nucleic acids, act as the common energy carrier of the cell and play a major role as regulatory molecules. While the intermediate and enzymatic steps of the purine biosynthetic pathway have been determined, the mechanism of regulation is not known. The purpose of this proposed research is to investigate the nature of the regulation of this major biosynthetic pathway. A method for the systematic isolation of regulatory mutants is proposed. This involves the isolation of gene fusions which places the lactose structural genes under the control of various purine control regions. The employment of lac fusions has the added benefit of assaying beta-galactosidase in place of the normal purine enzymes as the substrates are not readily available and the available assays are somewhat insensitive. The pur-lac fusion strains will be employed for the systematic isolation of regulatory mutations affecting the purine biosynthetic pathway. These mutants can reasonably be expected to fall into two classes; cis acting and trans acting. Specialized lambda transducing phages and/or plasmids will be isolated which carries the individual pur-lac fusions. Recombinant DNA techniques will be utilized for further restriction analysis and physical characterization of the purine control region DNA. The purine nucleotide pool levels will be determined by HPLC and correlated with the enzyme levels of the purine biosynthetic pathway in both wild type strains and regulatory mutants.